1. Field of the Invention
The present invention relates generally to the field of molecular biology. More particularly, it concerns the discovery of a DNA fragmentation factor that triggers nuclear changes during apoptosis. Methods and compositions for making and using the same are disclosed.
2. Description of Related Art
Apoptosis, or programmed cell death, is executed through a xe2x80x9csuicidexe2x80x9d program that is built into all animal cells (reviewed by White, 1996; Wyllie, 1995). Cells undergoing apoptosis show distinctive morphological changes, including membrane blebbing, cytoplasmic and nuclear condensation, chromatin aggregation and formation of apoptotic bodies (Wyllie, 1980). The biochemical hallmark of apoptosis is the cleavage of chromatin into nucleosomal fragments (Wyllie et al., 1980). Multiple lines of evidence indicate that apoptosis can be triggered by the activation of a family of cysteine proteases with specificity for aspartic acid residues, including CED-3 of C. elegans, CPP32/Yama/Apopain of humans, and DCP-1 of Drosophila (Yuan et al., 1993; Xue et al., 1996; Femandes-Alnemri, et al., 1994; Tewari, et al., 1995; Nicholson, et al., 1995; Song, et al., 1997). Recently, these proteins have been designated as caspases (Alnemri et al., 1996).
The most intensively studied apoptotic caspase is caspase-3, previously called CPP32/Yama/Apopain (Fernandes-Alnemri, et al., 1994; Tewari, et al., 1995; Nicholson, et al., 1995). Caspase-3 normally exists in the cytosolic fraction of cells as an inactive precursor that is activated proteolytically when cells are signaled to undergo apoptosis (Schlegel et al., 1996; Wang et al., 1996). Multiple apoptotic signals, including serum withdrawal, activation of Fas, treatment with granzyme B, ionizing radiation, and a variety of pharmacological agents, activate caspase-3 (Chinnaiyan et al., 1996; Darmon, et al., 1996; Datta et al., 1996, I997; Erhardt and Cooper, 1996; Hasegawa et al., 1996; Jacobsen et al., 1996; Martin et al., 1996; Schlegel et al, 1996).
A caspase-3-specific tetrapeptide inhibitor, Ac-DEVD-CHO, can abolish the ability of cytosol from apoptotic cells to induce apoptosis in normal nuclei and block the initiation of the cellular apoptotic program m response to apoptotic stimuli (Nicholson et al., 1995; Dubrez, et al., 1996; Jacobsen et al., 1996). Deletion of caspase-3 from the mouse genome through homologous recombination results in excessive accumulation of neuronal cells, owing to a lack of apoptosis in the brain (Kuida et al., 1996). Addition of active caspase-3 to normal cytosol activates the apoptotic program (Enari et al., 1996). These data indicate that caspase-3 is both necessary and sufficient to trigger apoptosis.
The identified substrates of caspase-3 include poly(ADP-ribose) polymerase (PARP) (Tewari et al., 1995; Nicholson et al., 1995), sterol-regulatory element binding proteins (SREBPs) (Wang et al., 1995; 1996), the U1 associated 70 kDa protein (Caciola-Rosen et al., 1996), D4GDI (Na et al., 1996), huntingtin (Goldberg et al., 1996), and the DNA-dependent protein Kinase (Casciola-Rosen et al., 1996; Song et al., 1996). It is not known whether the cleavage of any of these substrates plays a causal role in apoptosis.
Given that apoptosis is tightly regulated and has been linked to pathways that are dysregulated in a variety of diseases including cancer, it is important to identify mechanisms by which to control this process.
The present invention is directed towards the identification of factors involved in apoptosis. Thus in a preferred embodiment, there is provided an isolated polypeptide encoding a DFF40 DNA fragmentation factor. In particularly preferred embodiment, the DNA fragmentation factor has the amino acid sequence as set forth in SEQ ID NO:2. Also provided by the present invention is an isolated peptide having between about 10 and about 50 consecutive residues of a DFF40 DNA fragmentation factor. In certain defined aspects, the DNA fragmentation factor has an amino acid sequence of about 10 to about 50 consecutive residues of SEQ ID NO:2. In particular embodiments, the peptide is conjugated to a carrier molecule. In preferred embodiments, the carrier molecule is selected from the group consisting of KLH and BSA.
The present invention further provides a monoclonal antibody that binds immunologically to a DFF40 DNA fragmentation factor. In certain embodiments, the antibody does not bind immunologically to other human polypeptides. In particular embodiments, the antibody further comprises a detectable label. The detectable label may be selected from the group consisting of a fluorescent label, a chemiluminescent label, a radiolabel and an enzyme.
The present invention contemplates a hybridoma cell that produces a monoclonal antibody that binds immunologically to a DFF40 DNA fragmentation factor. In preferred aspects the antibody does not bind immunologically to other human polypeptides.
Also contemplated is a polyclonal antisera, antibodies of which bind immunologically to a DFF40 DNA fragmentation factor. In defined embodiments, the antisera may be derived from an animal other than a human.
Also provided by the present invention is an isolated nucleic acid comprising a region, or the complement thereof, encoding a DFF40 DNA fragmentation factor or an allelic variant thereof. In a preferred embodiment, the DFF40 DNA fragmentation factor is human. In other preferred embodiments, the DNA fragmentation factor has the amino acid sequence of SEQ ID NO:2. In other preferred embodiments, the nucleic acid sequence comprises the coding region having the sequence of SEQ ID NO:1 or the complement thereof. It is contemplated that the nucleic acid may be selected from the group consisting of genomic DNA, complementary DNA and RNA.
In particularly preferred embodiments, the nucleic acid is a complementary DNA and further comprises a promoter operably linked to the region, or the complement thereof, encoding the DNA fragmentation factor. In further embodiments, the nucleic acid further comprises a polyadenylation signal operably linked to the region encoding the DNA fragmentation factor. In still further embodiments, the nucleic acid farther comprises an origin of replication. In other aspects of this embodiment, the nucleic acid may be defined as a viral vector selected from the group consisting of retrovirus, adenovirus, herpesvirus, vaccinia virus and adeno-associated virus. In particularly preferred embodiments, the nucleic acid is packaged in a virus particle. In other alternative embodiments, the nucleic acid is packaged in a liposome.
The present invention further provides an isolated oligonucleotide of between about 15 and about 50 consecutive bases of a nucleic acid, or complement thereof, encoding a DFF40 DNA fragmentation factor. In preferred embodiments, the DNA fragmentation factor is human. In particularly preferred embodiments, the nucleic acid is the coding region of SEQ ID NO:1. In other preferred embodiments, the oligonucleotide is about 15 bases in length, is about 17 bases in length is about 20 bases in length is about 25 bases in length, is about 50 bases in length. Of course longer oligonucleotides also are contemplated.
Another aspect of the present invention provides a plasmid construct comprising a first nucleic acid encoding a DFF40 DNA fragmentation factor. In particular embodiments, the construct further comprises a first promoter active in eukaryotic cells positioned 5xe2x80x2 to the first nucleic acid. In yet additional embodiments, the construct, further comprises a second nucleic acid encoding a DFF45 DNA fragmentation factor. In yet another embodiment the construct further comprises an internal ribosome entry site (IRES), wherein the IRES is positioned 3xe2x80x2 to the upstream nucleic acid and 5xe2x80x2 to the downstream nucleic acid. In other embodiments, the construct further comprises a second promoter functional in eukaryotic cells, wherein the second promoter is positioned 5xe2x80x2 to the second nucleic acid.
It is contemplated that any promoter disclosed herein may be used, in particularly preferred aspects, the first promoter is selected from the group consisting of CMV IE, SV40 IE, RSV, xcex2-actin, tetracycline regulatable and ecdysone regulatable. In other embodiment the second promoter is selected from the group consisting of CMV IE, SV40 IE, RSV, xcex2-actin, tetracycline regulatable and ecdysone regulatable.
It is contemplated in certain embodiments of the present invention that the construct may further comprise a polyadenylation signal positioned 3xe2x80x2 to the first nucleic acid. In other embodiments, the expression construct comprising (i) a first polyadenylation signal positioned 3xe2x80x2 to the first nucleic acid and (ii) a second polyadenylation signal positioned 3xe2x80x2 to the second nucleic acid. In particularly defined aspects, the polyadenylation signal may be from BGH, thymidine kinase or SV40. In particular aspects the expression construct is a viral vector. In more defined embodiments, the viral vector is selected from the group consisting of retrovirus, adenovirus, vaccinia virus, herpesvirus and adeno-associated virus.
The present invention also provides a method of inducing apoptosis in a cell comprising the step of providing the cell with a DFF40 DNA fragmentation factor, wherein the provision of the DFF40 to the cell results in apoptosis. In certain embodiments, the DFF40 is provided as a protein complex comprising a DFF45 DNA fragmentation factor, and wherein the DFF45 is altered, with respect to wild-type DFF45, such that it lacks anti-apoptotic function but retains DFF40-chaperone function. In other embodiments, the DFF40 is provided as a protein complex comprising a DFF45 DNA fragmentation factor, and further comprising causing the DFF45 to be cleaved. In particular embodiments, the cleavage is effected by increasing the activity of caspase 3. In other embodiments, the providing comprises contacting the cell with a first expression construct comprising a first nucleic acid encoding a DFF40 polypeptide and a promoter functional in eukaryotic cells wherein the first nucleic acid is under the control of the promoter.
In particular aspects of the present invention, the method may further comprise providing a factor selected from the group consisting of a histone, a high mobility group protein and a nuclear factor. In certain aspects of the present invention, the expression construct further comprises a second nucleic acid encoding a DFF45 polypeptide, and wherein the DFF45 is altered, with respect to wild-type DFF45, such that it lacks anti-apoptotic function but retains DFF40-chaperone function. More particularly, the expression construct further comprises an internal ribosome entry site (IRES), wherein the IRES is positioned 3xe2x80x2 to the upstream nucleic acid and 5xe2x80x2 to the downstream nucleic acid. In preferred aspects, the expression construct further comprises a second promoter functional in eukaryotic cells, wherein the second nucleic acid is under the control of the second promoter. Additional aspects of this embodiment contemplate that the complex is encapsulated in a liposome.
In additional preferred aspects of this embodiments, the method may further comprise providing to the cells a second expression construct comprising a second nucleic acid encoding a DFF45 polypeptide and a second promoter functional in eukaryotic cells wherein the second nucleic acid is under the control of the second promoter. In particularly defined embodiment, the cell is a tumor cell. In other defined embodiments, the tumor cell may be derived from a tissue selected from the group consisting of brain, lung, liver, spleen, kidney, lymph node, small intestine, blood cells, pancreas, colon, stomach, breast, endometrium, prostate, testicle, ovary, skin, head and neck, esophagus, bone marrow and blood tissue.
The present invention further provides a method for inhibiting the growth of a cancer cell comprising the step of contacting a cancer cell with a DNA fragmentation factor designated DFF40 under conditions permitting the uptake of the DNA fragmentation factor by the cell, wherein the presence of the DFF40 in the cell induces apoptosis. In particular embodiments, the inhibition of growth may be measured by reduced proliferation, reduced cell migration, increase in contact inhibition, reduction in soft agar growth or restoration of cell cycling. In other embodiments, the cancer cell is within a subject. In other more preferred embodiments, the subject is a human.
It is contemplated that the DFF40 may be provided as a protein complex comprising a DFF45 DNA fragmentation factor, and wherein the DFF45 is altered, with respect to wild-type DFF45, such that it lacks anti-apoptotic function but retains DFF40-chaperone function. In other embodiments the DFF40 is provided as a protein complex comprising a DFF45 DNA fragmentation factor, and further comprising causing the DFF45 to be cleaved. In defined embodiments the cleavage is effected by increasing the activity of caspase 3. In certain embodiments, the providing comprises contacting the cell with a first expression construct comprising a first nucleic acid encoding a DFF40 polypeptide and a promoter functional in eukaryotic cells wherein the first nucleic acid is under the control of the promoter. In certain preferred aspects, the expression construct may further comprise a second nucleic acid encoding a DFF45 polypeptide, and wherein the DFF45 is altered, with respect to wild-type DFF45, such that it lacks anti-apoptotic function but retains DFF40-chaperone function.
Another aspect of the present invention provides a method for treating cancer comprising the step of contacting a tumor cell within a subject with a nucleic acid (i) encoding a DFF40 DNA fragmentation factor and (ii) a promoter active in the tumor cell, wherein the promoter is operably linked to the region encoding the DNA fragmentation factor, under conditions permitting the uptake of the nucleic acid by the tumor cell.
Also contemplated is a method of identifying a modulator of DFF40 activity comprising the steps of providing a cell expressing a DFF40/DFF45 complex; contacting the cell with a candidate substance; activating DFF40; and comparing the apoptosis of the cell in step (iii) with the apoptosis observed when the candidate substance is not added, wherein an alteration in apoptosis indicates that the candidate substance is a modulator the apoptotic activity. The cell may be a tumor cell. In particular embodiments, the apoptosis is measured using a parameter selected from the group consisting of DNA fragmentation, DNA condensation, DFF40 expression, nuclease activation, caspase activation, and DFF cleavage.
It is contemplated that the candidate substance independently may be a chemotherapeutic or radiotherapeutic agent. The candidate substance may also be selected from a small molecule library. In other embodiments, the candidate substance is a protein. In still further embodiment the candidate substance is a DFF45 analogue. In other embodiments the candidate substance may be a high mobility group (HMG) protein analogue. In defined aspects the HMG protein independently may be HMG-1, HMG-2, HMG-14 or an analogue thereof. In other embodiments the candidate substance is a nuclear factor. It is also contemplated that the candidate substance is an histone.
The present invention further contemplates a modulator of apoptotic activity identified according to a method comprising the steps of (i) providing a cell expressing a DFF401/DFF45 complex; (ii) contacting the cell with a candidate substance; (iii) activating DFF40; and (iv) comparing the apoptosis of the cell in step (iii) with the apoptosis observed when the candidate substance is not added, wherein an alteration in apoptosis indicates that the candidate substance is a modulator the apoptotic activity.
Also contemplated is an isolated DNA fragmentation factor complex for regulating chromatin stability, the complex comprising a DFF40 polypeptide and a DFF45 polypeptide. In preferred embodiment, the DFF40 subunit has the sequence of as set forth in SEQ ID NO:2. In other embodiments the DFF45 subunit has the sequence of as set forth in SEQ ID NO:4.
Another embodiments of this invention contemplates a method of producing a functional DNA fragmentation factor comprising providing to a cell a first nucleic acid encoding a DFF40 polypeptide; and a second nucleic acid encoding a DFF45 polypeptide; and expressing the complex in a cell, wherein the coexpression of the polypeptides allows for the formation of a functional DFF40 polypeptide. It is contemplated that the method may further comprise the step of causing the DFF45 polypeptide to be cleaved by caspase 3. In preferred embodiments, the first and the second nucleic acids are contained in the same expression construct and both are under the control a first promoter. In denied aspects the first and the second nucleic acids are contained in different expression constructs and are under the control a first and a second promoter, respectively. In additional embodiments, the second nucleic acid encodes a DFF45 polypeptide that is altered, with respect to wild-type DFF45, such that it lacks anti-apoptotic function but retains DFF40-chaperone function.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.